Southampton 0 Norwich City 1
John blew the chance to earn Southampton a much-needed point when he shot over in the 69th minute, after Gary Doherty had needlessly handled substitute Adam Hammill's cross.
John had only been given the penalty-taker's job after Grzegorz Rasiak missed from the spot in Saturday's 2-0 FA Cup win over Bury.
Unlike Poland striker Raziak, John did not even have the solace of firing home the rebound.
Southampton again had caretaker managers John Gorman and Jason Dodd in charge as their search for new Scotland coach Burley's successor goes on.
The duo can count themselves unlucky not to be celebrating two wins out of two after Saints were denied by the woodwork on no fewer than four occasions.
On-loan Manchester City striker Ched Evens compounded the home side's woes with his fourth goal of the season on the stroke of half-time.
Midfielder Lee Croft whipped in a dangerous cross from the right for Wales Under-21 international Evans to fire home with a fierce volley from the edge of the area.
Raziak set the tone for a frustrating evening for Southampton and the majority of a paltry crowd of just 18,004, when he shot against a post in the seventh minute.
Midfielder Rudi Skacel also found the woodwork when he smashed a 25-yard free kick against the top of Norwich's charmed crossbar and when defender Andrew Davies also failed to beat the upright with a hooked 45th minute shot, Gorman and Burley must have feared it was not their night.
Raziak was thwarted by the post for a second time four minutes into the second half after combining neatly with Andrew Surman.
John's hapless penalty compounded a miserable night for the home team, who start interviews to find their eighth manager in eight years in the morning.